The mammalian, including human, central nervous system (CNS, brain and spinal cord) has an extremely low ability for spontaneous anatomical and functional recovery after injury. This inability of the CNS to regenerate is due to a lack of a natural way to replace lost neurons and to establish intense functional connections between different neuronal populations after trauma. It has been demonstrated that central nervous system contains multipotent progenitor cells. These multipotent cells proliferate and differentiate into neurons, astrocytes and oligodendrocytes in vitro and in vivo. Progenitor cells or neuronal derivatives of these cells can be used for transplantation to stimulate anatomical and functional regeneration. Cell replacement and neuronal circuitry reconstruction strategies in human neurological conditions require a well-established source of neuronal cells. Techniques have been developed to isolate, propagate and differentiate neuronal stem cells from the fetal and adult human central nervous system. Unfortunately, propagation of these stem cells is time consuming (many months) and during propagation many cells loose their multipotency to differentiate in variety of neuronal types.
One of the crucial problems in cell therapy using autologous transplantable cells is propagation of neural stem cells in conditions that will result in a large number of multipotent cells whereas cells maintain the capacity to differentiate into variety of neural cell types. Numerous data clearly show that there is a balance between multipotent neural stem cell population and populations of neural progenitor cells (NPC) that are committed to certain differentiation pathway both in vivo and in vitro.
With regard to development, it is well established that different signaling routes (Wnt, Shh and BMP/TGFbeta, Notch and TK signaling cascades) are critical for proper gene expression at appropriate times since antagonistics biological processes, such as proliferation-differentiation and survival-apoptosis, are all integrated in the formation of a three-dimensional nervous (brain) tissue, whose function changes with time. In embryonic stages, the growth and proper development of nervous structures is dependent on the interaction between the glial cells and neurons and require the concerted actions of various bioactive peptides and hormone-like substances, and cell-cell and cell-substrate interactions. The necessity for these complex structural and hormonal interactions provide a challenge for the development of in vitro cell culture models that more accurately mimic the developing nervous system.
Similarly, the mammalian central nervous system (CNS; brain and spinal cord) has a limited ability for spontaneous recovery following an injury. This inability of the CNS to regenerate is caused by the lack of a natural pathway to replace lost neurons and re-establish the functional connections between different neuronal populations after trauma.
However, it has been demonstrated that the CNS contains multipotent progenitor cells (nervous system-derived progenitor and stem cells; NSC's) that can proliferate and differentiate into neurons, astrocytes and oligodendrocytes both in vitro and in vivo. Progenitor cells (or neuronal derivatives of these cells) can be transplanted to stimulate anatomical and functional regeneration. Techniques have been developed to isolate, propagate and differentiate neuronal stem cells from the CNS, but propagation of these stem cells is time consuming (typically many months) and many cells lose their multipotency to differentiate as a result of the process.
Current culture models using continuous exposure of cultured NSC's to high levels of bFGF (basic Fibroblast Growth Factor), LIF (Leukemia Inhibitory Factor) and EGF (Epidermal Growth Factor) have not yielded substantial numbers of human NSC's (hNSC's) in vitro, thus preventing the use of propagated hNSCs for autologous transplantation purposes. Therefore, there exists a need for methods of expansion of hNSC's into neuronal cells.